Comparison of two methods of preparing enzyme-antibody conjugates: Application of these conjugates for enzyme immunoassay

Abstract

Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The first procedure utilizes the homobifunctional crosslinking reagent N,N′-o-phenylenedimaleimide. Sulfydryl residues were introduced into second antibodies by reaction with methyl-mercaptobutyrimidate. The modified antibodies were reacted with N,N′-o-phenylenedimaleimide, excess reagent was removed by gel filtration, and the activated antibodies were cross-linked to β-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The second method utilizes the heterobifunctional meta-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and the activated antibodies were crosslinked to β-galactosidase. Typically, approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these two procedures were used as labels in an immunoassay system and were able to detect approximately 5 to 10 ng of first antibodies. The MBS procedure was simpler to perform, could more easily be adapted to large-scale work, and gave more reproducible results, and the conjugates produced were able to detect slightly lower concentrations of first antibody.